Protozoan parasites that belong to the genus Plasmodium, are the causative agent for malaria, and are estimated to be responsible for 200-500 million new cases of malaria each year. The parasite has a complex life cycle that involves both extracellular and intracellular forms. Infection is initiated in the host when an anopheline mosquito injects sporozoites during a blood meal feeding. The sporozoites then travel through the vascular system to the liver and invade hepatocytes, thus initiating the exoerythrocytic (EE) stage. Each uninuclear parasite subsequently undergoes division and differentiation to form a mature liver stage schizont with thousand to tens of thousands of uninuclear merozoites. The time for development in the liver stage schizont varies with the species, e.g., 5-7 days for Plasmodium falciparum. 
At the conclusion of the initial liver stage, thousands of merozoites are released into the bloodstream where they invade erythrocytes. An erythrocytic stage (or “red cell” stage) cycle of 48-72 hr, depending upon the species, then ensues. Some red cell stages will differentiate into male and female gametocytes. Once ingested by a mosquito they can combine and form a zygote, which, upon further development, can produce sporozoites. These sporozoites are later inoculated by the mosquito into another host, thus repeating the cycle.
The sporozoites that are injected by the bite of female Anopheles mosquitoes rapidly reach the liver sinusoids. Contrary to earlier assumptions, it is now believed that the numbers of inoculated sporozoites are small, about 10-100 per bite. Whether sporozoites directly penetrate hepatocytes or first travel through endothelial or Kupffer cells lining the sinusoid is still a topic of debate. However, in vitro invasion of hepatic cells suggests that passage through Kupffer cells is not an absolute requirement for EE development. Additionally, evidence from studies done on rats depleted of Kupffer cells has demonstrated that relative to intact animals, Kupffer cell depleted animals had a significant increase in the number of EE stage parasites. This strongly suggests that Kupffer cells are involved in the clearance of sporozoites, not in the facilitation of sporozoite invasion.
Because sporozoites only develop in hepatocytes, it is likely that invasion is mediated by specific interactions between sporozoite proteins and hepatocyte receptors. Sporozoites appear to invade apically by invagination of the hepatocyte cell membrane which forms the parasitophorous vacuole membrane (PVM) surrounding the developing liver stage or exoerythrocytic (EE) parasite. As during merozoite invasion of erythrocytes, material appears to be secreted from sporozoite rhoptries during invasion. Shortly after invading a hepatocyte, the inner membrane and subpellicular microtubules of the thin sporozoite (1.5×10-20 μm) break down, and this area bulges out creating a uninucleated 3-5 μm trophozoite bounded by a plasma membrane situated in a vacuole surrounded by a PVM. The early exoerythrocytic stage parasite develops into a spherical, mature liver stage schizont that occupies most of the volume of the hepatocyte.
During schizogony, parasite antigens are inserted into the PVM which can form deep invaginations into the infected hepatocyte, particularly next to the hepatocyte nucleus. Development of mature EE schizonts containing at least 2,000 uninucleated merozoites takes 42-48 hours for the rodent malarias P. yoelii and P. berghei. P. falciparum liver stage development takes 5-7days forming up to 30,000 merozoites within a 80-100 μm schizont. During P. vivax EE development, some trophozoites do not develop further and may persist as small hypnozoites within hepatocytes for several years. It has been proposed that relapses of P. vivax arise from hypnozoites that are triggered to develop by unknown mechanisms. The signals and parasite molecules responsible for the development of a uninucleate sporozoite to a fully mature liver stage schizont and for rupture of this schizont are unknown.
Plasmodium-infected hepatocytes have been demonstrated to be an important target of a protective immune response in rodent models of malarial infection. This finding makes the identification and characterization of plasmodial antigens expressed in the infected hepatocyte or exoerythrocytic (EE) stage of the parasite and the immune responses against these antigens crucial for the development of a pre-erythrocytic stage malaria vaccine. A source of EE stage parasites is vital for the selection and characterization of monoclonal antibodies, that identify EE stage antigens, and to characterize the expression of Plasmodium genes expressed in infected hepatocytes.
Unfortunately, it is difficult to obtain biological material containing the EE forms of P. falciparum. Small quantities of P. falciparum EE stage parasites have been produced in primary human hepatocyte cultures and, in a single human hepatoma cell line. The human cell line is difficult to work with and the scarcity of available human liver tissue and low infection rates obtained in the in vitro systems make these approaches less attractive. While mouse forms of malaria exist, P. falciparum schizonts develop only in hepatocytes of human, chimpanzee or to a much lower degree in some non-human primates. Chimpanzees have been infected with parasites and liver tissue obtained, by biopsy, for cryosectioning. However, work involving chimpanzees is quite expensive, and does not lend itself to anything more complex than limited tissue harvesting.
There is a need in the field for a reliable small animal model for P. falciparum infection. The present invention addresses this need.